Threonine synthase from Lemma was purified some 50-fold, and separated from cystathionine Gamma-synthase. Threonine synthase showed a high specificity for the carbon substrate (O-phosphohomoserine) and the nucleophile (hydroxyl ion); nucleophilic attack by hydroxyl ion was restricted to carbon 3 of O-phosphohomoserine and was stereospecific. No evidence was obtained for major feedback inhibition, repression or covalent modification of the enzyme by threonine and/or isoleucine. Preliminary experiments indicate that two unexpected properties may be of regulatory significance. The first is that threonine synthase activity in gel-filtered extracts was decreased approximately 50% by growth of plants with methionine, and increased in plants limited in methionine by growth with lysine + threonine. These observations suggest possible down-regulation of threonine synthase by excess methionine. The second unexpected property is that threonine synthase was inhibited (approx. 70%) by AMP (67 MuM) or Pi (1mM), while cystathionine Gamma-synthase was relatively insensitive to these compounds. Inhibitions comparable to those with AMP or Pi were not obtained with a variety of other nucleotides or anions (pyrophosphate, sulfate, chloride). These findings raise the possibility that the physiological concentrations of AMP (20 MuM) and Pi (14 mM) in Lemna may inhibit the activity of threonine synthase relative to that of cystathionine Gamma-synthase, and thereby affect the balance of fluxes into the threonine and methionine biosynthetic branches. Preliminary results indicate that 5 feet-chloroadenosine (Ch1Ado) is an effective inhibitor of the conversion of methylthioadeniosine (MTA) to methionine. Ch1Ado prevents MTA from relieving the severe growth inhibition caused by the combined presence of propargylglycine and lysine + threonine; the same concentration of Ch1Ado alone had no effect on growth. These experiments suggest that conversion of MTA to methionine is probably not an absolute requirement for normal growth on Lemna, but becomes crucial when net synthesis of methionine via transsulfuration is inhibited e.g. by propargylglycine and lysine + threonine.